Abstract
In practical production, almost all rams and about 50% of ewes are used
to fatten. Researchers have proved that ewe ovariectomy could improve
the productivity significantly, but the specific molecular mechanism is
still unknown. In this study, five independent cDNA libraries (three
and two from ovariectomized and normal ewe longissimus dorsi samples,
respectively) were constructed to thoroughly explore the global
transcriptome, further to reveal how the ovariectomized ewes influence
muscle development by Illumina2000 sequencing technology. As a result,
205 358 transcripts and 118 264 unigenes were generated. 15 490 simple
sequence repeats (SSRs) were revealed and divided into six types, and
the short repeat sequence SSR (monomers, dimers, trimers) was the
domain type. Single nucleotide polymorphism analysis found that the
number of transition was greater than the number of transversion among
the five libraries. Furthermore, 1612 differently expressed genes
(DEGs) (Log2fold_change > 1 and p < 0.05) were revealed between
ovariectomized and normal ewe groups, in which 903 genes were expressed
commonly in the two groups, and 288 and 421 genes were uniquely
expressed in normal and ovariectomized ewe groups, respectively. Gene
Ontology (GO) analysis categorized all unigenes into 555 GO terms and
56 DEGs were significantly categorized into 43 GO terms (p < 0.05).
KEGG enrichment analysis annotated 12 976 genes (containing 137 DEGs)
to 86 pathways, among them 24 and 11 DEGs involved in development and
reproduction associated pathways, respectively. To validate the
reliability of the RNA-seq analysis, 22 candidate DEGs were randomly
selected to perform quantitative real-time polymerase chain reaction.
The result showed that 9 and 1 genes were significantly and
approximately significantly expressed in control and treatment group,
respectively, and the results of RNA-seq are believable in this study.
Overall, these results were helpful for elucidating the molecular
mechanism of muscle development of ovariectomized animals and the
application of female ovariectomy in fattening.
Keywords: transcriptome, ovariectomy, goats, muscle, development,
growth
1. Introduction
With the improvement of living standards and the demand for mutton, in
practical production, as much as 50% of ewes still are used to fatten.
Ram castration has been applied in production and achieved effective
results [[41]1,[42]2]. Many researchers have proved that ewe
ovariectomy (removal of the ovary) could improve the productivity
significantly like ram castration. Study on mice found that the
ovariectomized groups showed a greater body mass than the group without
ovariectomy, no matter fed on standard chow or high-fat diet [[43]3].
Studies on goat found that meat performance and muscle tissue nutrient
content of the ewe kid could be improved by ovariectomy [[44]4,[45]5].
Zhang et al. slaughtered 18 goats (nine goats in ovariectomy group and
nine goats in control group) on day 50 after ovariectomization, and
measured meat performance. They found that the average daily gain, live
weight, carcass weight, net meat mass, and loin eye area of
ovariectomized goats were significantly higher than those of the
control goats (p < 0.05). Meanwhile, an average loss of bone weight
(0.40 kg) was observed in the ovariectomized goats compared with the
unovariectomized goats (p < 0.05). But there was no significant
difference between the ovariectomized group and control group in fat
weight (p > 0.05) [[46]5]. Although these findings proved that ewe
ovariectomy could improve the productivity significantly, the specific
molecular mechanism is still unknown. Thus it is meaningful to reveal
the molecular mechanism of ewe ovariectomy improving the productivity
and to applied ovariectomy in practice production.
Ovariectomy has been used as effective disease treatment method and
breeding control in animals, such as pig, dog, rabbit, rat, even
hippopotamus [[47]6,[48]7]. Moreover, experimental studies often apply
ovariectomized animals to mimic menopause, because the characters of
ovariectomized animals resemble the metabolic alterations observed in
women [[49]8,[50]9]. Moreover, ovariectomized animals serving as
disease or medicine research models are widely adopted [[51]10,[52]11].
Ovariectomy-induced osteoporosis rabbit models have been used to
examine the effects of autologous dedifferentiated fat cell
transplantation on bone regeneration and investigate the effects of a
traditional Chinese herb product, OsteoKing, for the treatment of bone
disease [[53]12,[54]13]. Ovariectomized rat was chosen as the model to
analyse the influence of ovariectomy on alveolar bone and tibiae
[[55]14]. Thus ovariectomy is a mature technique, which can be applied
to goat or other livestock in practical production.
Boer goat is a world famous meat-type breed, which has great
reproductive capacity and growth rate. Also, Boer goat is one of the
most adaptable breeds to fit the natural ecological environment and it
has been spread to many countries [[56]15–[57]17]. Hybridization
experiment showed that Boer goat could improve the production
performance of local goat breed [[58]18]. Guanzhong dairy goat is a
well-known Chinese dairy goat breed, which is widely distributed in the
Guanzhong region in Shaanxi Province of China [[59]19]. The
introduction, breeding, and hybridization with Guanzhong dairy goat of
Boer goat have strongly promoted the development of mutton goat
industry in China. The hybridization test of Boer goat and Guanzhong
dairy goat started in 1995 in Shaanxi province, China. Early practical
test results showed that the F1 hybrid of Boer goat and Guanzhong dairy
goat has better growth rate than the F1 hybrid of Boer goat and other
goat breeds. And the F1 hybrid of Boer goat and Guanzhong dairy goat
could well adapt to the local ecology characteristic of Shaanxi
province. Nowadays, the F1 hybrid of Boer goat and Guanzhong dairy goat
is used for fattening in production practice.
Muscle is one of the main factors which influence the growth and
development of animals. Longissimus dorsi being muscle tissue, its
growth rate is relatively stable compared to the other muscles on other
body parts, such as leg muscle. The growth rate of longissimus dorsi is
affected by the growth characteristic of goat itself and the raising
level, but is less affected by exercise intensity. There are some
high-throughput sequencing of RNA (RNA-seq) studies that pay attention
to the growth and development of animals just detecting the mRNA or
miRNA expression levels in longissimus dorsi muscle [[60]20,[61]21].
According to the results, this study conjectured that ovariectomy might
increase the mRNA expression levels of some genes which promote muscle
growth and development.
RNA-seq is an efficient way to map and quantify transcriptome and to
analyse global gene expression changes in different samples
[[62]22–[63]24]. Thus this study regarded F1 hybrid of Boer goat and
Guanzhong dairy goat as model animal to thoroughly investigate the
global transcriptome of ovariectomized ewe and normal ewe longissimus
dorsi muscle samples by RNA-seq method. The objective of this study was
to clarify the molecular mechanism of ovariectomy affecting animal
growth, offer valuable resource for female animal fattening and provide
essential information for further research on muscle development of
ovariectomized animal.
2. Material and methods
2.1. Experimental design and sample collection
This study used six female Boer hybrid goats (Boer goat♂ × Guanzhong
dairy goat ♀) as models, which were chosen from national mutton
industrial technology system Baoji comprehensive experimental station,
Shaanxi Province, China. Six female hybrid kids with similar body
weight at about five months old were randomly divided into treatment
group (three kids) and control group (three kids). The goats in the
treatment group were ovariectomized at the beginning of the experiment
and the goats in control group were untreated. All the six goats were
slaughtered at the 50th day. The longissimus dorsi muscle samples were
collected immediately after slaughtering, and were snap-frozen in
liquid nitrogen, then stored at −80°C until RNA extraction [[64]25].
2.2. RNA extraction, library construction and sequencing
Total RNA was extracted from the six samples using Trizol reagent
(Takara, Dalian, China) according to the manufacturer's instructions.
The RNA purity was determined via the OD[260]/OD[280] ratio and
OD[260]/OD[230] ratio. The integrity was evaluated using gel
electrophoresis and checked by RNA integrity number (RIN) value. RNA
samples with a RIN value greater than 8.0 (8.0 out of 10.0),
OD[260]/OD[280] ratio greater than 1.9, OD[260]/OD[230] ratio no less
than 1.7 and the amount of RNA no less than 20 µg were selected for
deep sequencing [[65]26].
The RNA was treated with DNase I for 30 min at 37°C to remove residual
DNA. Then mRNA was enriched and purified using beads with oligo (dT)
[[66]27]. The purified mRNA was fragmented to approximately 350 nt
fragments using the RNA fragmentation kit. The first strand cDNA was
synthesized using purified RNA fragments as template and hexamer
primers. After the first strand was synthesized, the buffer
(Invitrogen, 20 µl), dNTPs (0.25 mM/μl), RNaseH (0.05 U/μl) and DNA
polymerase I (0.5 U/μl) were added to synthesize the second strand cDNA
[[67]26]. The short cDNA fragments were further purified using the
QIAQuick PCR extraction kit (LianChuan Sciences, Hangzhou, China). The
samples were then subjected to 3′ end repaired, a single ‘A’ base and
an adapter ligation. Agarose gel electrophoresis was used to filter the
suitably sized fragments (350 ± 50 bp), which would be used as the
templates for the polymerase chain reaction (PCR) amplification and
RNA-seq library construction. Then the sequencing of the libraries was
performed using Illumina HiSeq 2000 (LianChuan Sciences, Hangzhou,
China).
Taken together, there was one sample that did not meet the requirement.
Thus finally we constructed five libraries to investigate the global
transcriptome of ovariectomized ewes (treatment group: three samples)
and normal ewes (control group: two samples) longissimus dorsi muscle
samples by deep RNA sequencing method.
2.3. Transcript and unigene assembly and annotation
The valid data, which can be used for subsequent analysis, were
obtained by removing the adapters and low quality reads in raw reads.
The transcript and unigene assembly was carried out using short
assembly program Trinity method [[68]28,[69]29]. The assembled datasets
were deposited in a publicly available database: Gene Expression
Omnibus (GEO): [70]GSE84110:
[71]http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84110.
All assembled unigenes were compared with public database, NCBI
non-redundant protein sequences (NR), SWISS-PROTPROT, Kyoto
Encyclopedia of Genes and Genomes (KEGG), euKaryotic Ortholog Groups
(KOG) and Pfam to make functional annotation by similarity analysis.
Sequences alignment using BLAST software, only a significance threshold
of E-value < 10^−5 can be used as annotation information.
2.4. Identification of unigene simple sequence repeat and single nucleotide
polymorphism
Simple sequence repeat (SSR), also named microsatellite DNA, is
composed of many repetitive nucleotide sequences. The numbers of
repetitive nucleotide sequences in different individuals result in
polymorphism in group. The SSR in this study was detected by
MiscroSAtellite software using assembled unigenes as reference
sequences. Single nucleotide polymorphism (SNP) is similar to SSR,
which can be used for genetic diversity analysis, marker-assistant
selection, QTL analysis, genetic linkage map construction etc. This
study analysed the SNPs in gene coding sequences using Bowtie software,
and screened reliable SNP loci using Samtools software.
2.5. Differently expressed genes analysis
All reads were mapped onto the non-redundant set of transcripts to
quantify the abundance of assembled transcripts. The gene expression
level was computed by Bowtie 0.12.8 using single-end mapping method,
and further measured by the reads per kilobase of exon model per
million mapped reads value (RPKM), which can be calculated as follows:
RPKM = total exon reads/[mapped read (millions) * exon length (KB)]
[[72]30]. The high RPKM represents the high expression level of gene.
The Log2fold_change (calculated by log2 (sample_2 RPKM/sample_1 RPKM))
and P value were used to calculate the different expression between two
samples. False discovery rate (FDR) is correct for the p value
[[73]31]. When p < 0.05, higher values of Log2fold_change and lower FDR
showed more significant differently expressed genes (DEGs). Moreover,
the DEGs were subjected to Gene Ontology (GO) and KEGG analysis.
GO database, an international standard gene functional classification
system, was used to predict and illuminate the function of gene product
on molecular, biological process and cellular component [[74]32]. The
DEGs were mapped to the GO terms in the database
([75]http://www.geneontology.org/) firstly. Then the gene numbers in
every GO term were calculated and the significantly enriched GO terms
were found using corrected p ≤ 0.05 as a threshold. The p value was
calculated by the following formula:
[MATH: p=1−∑i=0
m−1MiN−Mm−iNn, :MATH]
where N is the number of all genes; n is the number of the DEGs in N; M
represents the number of the genes which were categorized to a certain
GO term; m represents the number of the DEGs which were categorized to
a certain GO term.
KEGG ([76]http://www.genome.jp/kegg/) was used to perform a pathway
enrichment analysis of unigenes [[77]33]. Pathway enrichment analysis
can confirm the main biochemical pathway and signal pathway which DEGs
participated in. The significantly enriched KEGG pathway using
corrected p ≤ 0.05 as a threshold. The calculation formula of the p
value was the same as that in the GO analysis, where N is the number of
all genes with pathway annotated; n is the number of the DEGs in N; M
is the number of the genes annotated to a certain pathway; m is the
number of the DEGs annotated to a certain pathway.
2.6. Experiment verification and statistical analysis
To quantitatively determine the reliability of our transcriptome data,
22 significantly DEGs were selected randomly from different pathways
and functional categories to test their expression levels via the
quantitative real-time PCR (qRT-PCR) method. Ribosomal protein L19
(RPL19), which is the most stable housekeeping gene in goat muscle
samples among PITX1, PITX2, GAPDH and beta-actin (unpublished data),
was used to normalize the expression of target genes in longissimus
dorsi muscle samples. Total RNA samples were reversed to cDNA using the
PrimeSript™ RT reagent kit (TaKaRa, Dalian, China) according to the
manufacturer's recommendations. qRT-PCR was performed using the SYBR^®
Premix Ex Taq™ kit (TaKaRa, Dalian, China) in a Bio-Rad CFX96 real-time
PCR system (Hercules, CA, USA). The primers used in this study are
shown in electronic supplementary material, table S1. The qRT-PCR
reaction system composition and program are the same as in a previous
study [[78]34]. Individual samples were run in triplicate.
Relative expression of mRNA was calculated using the 2^−ΔΔCt method
[[79]35]. Student t-test was used to analyse independent samples via
SPSS (version 17.0, SPSS, Inc., Chicago, IL, USA), and data were
expressed as the mean ± 1 standard deviation of duplicates. A p
value < 0.05 was considered statistically significant.
3. Results and discussion
3.1. De novo assembly and functional annotation
Five libraries were constructed to obtain a global view of the
transcriptome of normal ewes (control group: BZJ_1_N and BZJ_3_N) and
ovariectomized ewes (treatment group: BZJ_2_T, BZJ_4_T and BZJ_5_T)
longissimus dorsi muscle samples by deep RNA sequencing method. As a
result, we acquired an average 5.3 billion clean reads from each
library after filtering the redundant sequences. The lowest valid ratio
(reads) is as high as 99.81% (electronic supplementary material, table
S2).
Since the goat genome is not comprehensive enough, we used de novo
assembly method to construct transcripts and unigenes from RNA-seq
reads by Trinity [[80]28]. The high quality reads (Q > 20) were
assembled into transcripts and unigenes, with a fixed k-mer of 25
[[81]36,[82]37]. In total, 205 358 transcripts and 118 264 unigenes
were generated, with average lengths of 1291 bp and 919 bp,
respectively ([83]figure 1). All assembled unigenes were firstly
aligned to NR protein database, which contains the protein sequences
translated by all DNA sequences in GenBank and come from other
databases. According to the results of blast, 44 504 (37.63%) unigenes
obtained annotations in NR database, and they were similar to genes in
other species, such as Ovis aries (28%), Bos taurus (16.8%) and Bos
grunniens (8.5%) ([84]figure 2). To better understand the functionality
of these unigenes, they were further blast in other databases. The
annotation numbers and ratios of unigenes against various databases
were SWISS-PROTPROT 41 335 (34.95%), KEGG 39 389 (33.31%), KOG 38 284
(32.37%) and Pfam 31 321 (26.48%). The rate of the annotated genes in
this study was the same as that in another study of goat [[85]38].
Figure 1.
[86]Figure 1.
[87]Open in a new tab
The length distribution of transcript (a) and unigene (b). The
horizontal axis shows the length extent of transcript and unigene, and
the vertical scale shows the numbers of transcript and unigene in the
sequence data.
Figure 2.
[88]Figure 2.
[89]Open in a new tab
Unigenes distribution in NCBI non-redundant protein sequences database.
3.2. The distribution of unigene simple sequence repeat and single nucleotide
polymorphism
After SSR analysis, a total of 15 490 SSRs were revealed, and they were
divided into six types according to the length of the repeat sequence.
The numbers for each type were monomers (7628), dimers (3832), trimers
(3474), quadmers (202), pentamers (279) and hexamers (75) ([90]figure
3). Among these, the short repeat sequence SSR (repeat nucleotide ≤ 3)
was the domain type. The SNP analysis found 84 055 (BZJ_1_N), 84 055
(BZJ_2_T), 70 044 (BZJ_3_N), 70 044 (BZJ_4_T) and 58 575 (BZJ_5_T) SNPs
in the transcriptome alignment file of the five samples. The
transitions were more than the transversions among the five libraries.
The numbers of different SNP types in the five libraries are shown in
[91]figure 4. SSR and SNP molecular markers are the basis for genetic
mapping and comparative genomic analysis. Although some SSR markers
have been researched in goat, few studies have investigated the SSRs on
mRNA [[92]19]. The distribution of the SSRs and SNPs revealed in this
study in the genome was consistent with other studies in goat [[93]38].
The results confirmed that RNA-seq is an efficient method to uncover
genetic variations in transcribed regions [[94]39].
Figure 3.
[95]Figure 3.
[96]Open in a new tab
The SSR numbers of each SSR type.
Figure 4.
[97]Figure 4.
[98]Open in a new tab
The summary of the numbers of different single nucleotide polymorphism
(SNP).
3.3. Differentially expressed genes analysis
The transcript expression levels of the five samples were calculated by
RPKM values and exhibited using box plot ([99]figure 5). Furthermore, a
total of 1612 (Log2fold_change > 1 and p < 0.05) and 216
(Log2fold_change > 1 and p < 0.01) DEGs were revealed between control
group and treatment group longissimus dorsi muscle samples (electronic
supplementary material, table S3). In 1612 DEGs, 718 genes were
upregulated and 894 gene were downregulated in longissimus dorsi muscle
samples of ovariectomized ewes. Meantime, 903 genes were expressed
commonly in the two groups, 288 and 421 genes were uniquely expressed
in control and treatment group, respectively ([100]figure 6). Volcano
plots could infer the whole pattern of the DEGs and determine any
systematic bias that may be present between conditions from the levels
of log2 fold change and −log10 p value. Log2 fold change represented
the change of genes expression multiple between samples. And −log10 p
value represented the statistically significant DEGs between samples.
Thus we used the volcano plots to explore the relationship between the
fold change and the significance ([101]figure 7).
Figure 5.
Figure 5.
[102]Open in a new tab
Expression levels of the five samples. The vertical scale shows the
RPKM values of each sample. Every box region represents five
statistics, which, from top to bottom are, in order, maximum, 3rd
quartile, median, 1st quartile and minimum.
Figure 6.
Figure 6.
[103]Open in a new tab
The differentially expressed genes that are uniquely or commonly
expressed in control group (normal ewes) and treatment group
(ovariectomized ewes) longissimus dorsi muscle samples. The numbers in
each section indicate the number of differentially expressed genes.
Figure 7.
[104]Figure 7.
[105]Open in a new tab
Volcano plots of the differently expressed genes in control group
(normal ewes) and treatment group (ovariectomized ewes) longissimus
dorsi muscle samples. The blue plots represent the significance; the
red plots represent no significance; log2 fold change is equal to log2
(treatment group value/control group value).
To investigate the potential function of the DEGs, GO analysis was
performed to categorize DEGs into different biological function class.
All unigenes (including 1612 DEGs) were categorized into 555 GO terms
in the GO database. But only 56 DEGs were significantly categorized
into 43 GO terms (p < 0.05) (electronic supplementary material, table
S4). The other 1556 DEGs were also categorized into the corresponding
GO terms, but did not reach the significant level. These 56 DEGs were
further categorized into three categories: biological process (34
DEGs), molecular function (14 DEGs), and cellular component (8 DEGs)
([106]figure 8). In the cellular component category, 2 DEGs were
significantly enriched in the neuromuscular junction class
(p = 0.0173). In the biological process, 2 DEGs were enriched in the
synapse assembly class (p = 0.0037); and in molecular function classes,
2 DEGs were enriched in the sulfiredoxin activity class (p = 0.002).
All the GO analysis information is arranged in electronic supplementary
material, table S4.
Figure 8.
[107]Figure 8.
[108]Open in a new tab
Significantly enriched GO terms of differently expressed genes in the
analysed goat longissimus dorsi muscle samples.
To further identify the active biochemical pathways of unigenes, genes
were mapped to the KEGG database, which can be used to systematically
analyse the functions and network of genes. Among them, a total of
12 976 genes (containing 137 DEGs) were annotated to 86 pathways in
KEGG database, but no significance (p > 0.05). KEGG enrichment analysis
found 24 and 11 differentially expressed genes involved in development
and reproduction associated pathways, respectively. For example,
laminin alpha 1 (LAMA1) is essential to form and maintain muscle
integrity [[109]40]. For Bardet–Biedl syndrome 10 (BBS10), its mutation
could cause Bardet–Biedl syndrome, which includes diabetes and obesity
[[110]41]. The paternally expressed 10 gene (PEG10) plays important
roles in cell proliferation, apoptosis and meat quality traits
[[111]42,[112]43]. Histone deacetylase 9 (HDAC9) can promote cell
proliferation, regulate cell cycle progression and inhibit apoptosis
[[113]44,[114]45]. These results are listed in [115]table 1, and all
the pathways and related information are listed in electronic
supplementary material, table S5. Although no significance was found in
KEGG enrichment analysis, the KEGG analysed results were a good
indication of the function of the genes which were enriched in specific
pathways [[116]29,[117]37]. The above four genes, LAMA1, BBS10, PEG10
and HDAC9, were all enriched in the right pathways, which was
consistent with their functions (electronic supplementary material,
table S5). This means that the KEGG analysis results could be used as
reference to search the functions of new genes and reveal important
genes.
Table 1.
Differentially expressed genes involved in development and reproduction
associated pathways.
no. function^a development associated pathways unigenes^b
1 development apoptosis 172(1)
2 development regulation of actin cytoskeleton 352(3)
3 development amyotrophic lateral sclerosis (ALS) 35(1)
4 development MAPK signalling pathway, yeast 20(1)
5 development Jak-STAT signalling pathway 159(3)
6 development inositol phosphate metabolism 152(2)
7 development ubiquitin mediated proteolysis 116(1)
8 development MAPK signalling pathway 426(5)
9 development hypertrophic cardiomyopathy (HCM) 181(1)
10 development mTOR signalling pathway 119(2)
11 development cardiac muscle contraction 187(2)
12 development insulin signalling pathway 181(1)
13 development Wnt signalling pathway 221(1)
14 reproduction steroid hormone biosynthesis 13(1)
15 reproduction endometrial cancer 127(2)
16 reproduction ErbB signalling pathway 200(2)
17 reproduction aldosterone-regulated sodium reabsorption 56(1)
18 reproduction oocyte meiosis 230(1)
19 reproduction progesterone-mediated oocyte maturation 117(2)
20 reproduction GnRH signalling pathway 199(2)
[118]Open in a new tab
^aThe functions of the pathways are development or reproduction
associated.
^bRefers to the numbers of total unigenes and differently expressed
unigenes (in parentheses) mapped to the specific pathways.
3.4. Quantitative real-time polymerase chain reaction validation of the
candidate differently expressed genes
To validate the reliability of the RNA-seq analysis, 22 candidate DEGs
from different GO terms and KEGG pathways were successfully selected to
perform qRT-PCR. A heat-map was produced to explore the difference
expression of these 22 genes based on the RPKM values in RNA-seq
([119]figure 9). The candidate DEG sequences were blasted to the
reference RNA sequences in the National Center for Biotechnology
Information (NCBI) website. The result of qRT-PCR showed that the
expression changes of these 22 candidate DEGs in normal ewes and
ovariectomized ewes longissimus dorsi muscle samples had the same trend
as the results of RNA-seq. And among these 22 candidate DEGs, 9 and 1
genes were significantly and approximately significantly expressed in
control and treatment group, respectively ([120]figure 10). In
addition, there were two upregulated and seven downregulated genes
significantly differently expressed in ovariectomized group
([121]figure 10). Considering the differences of the experimental
methods, data calculation methods and statistical model, differences of
expression levels within a certain range are inevitable and acceptable.
Thus these results suggested that the result of RNA-seq is believable
in this study.
Figure. 9.
[122]Figure. 9.
[123]Open in a new tab
A heat-map exploring the differences in qRT-PCR detected genes
expression between samples. Different colours represent different
expression levels; the grey colour represents no expression. Control
group: BZJ_1_N and BZJ_3_N; treatment group: BZJ_2_T, BZJ_4_T and
BZJ_5_T.
Figure 10.
[124]Figure 10.
[125]Open in a new tab
Quantification of the mRNA levels of differently expressed genes. The
error bars are the s.e. of duplication. * and ** represent the
significantly different expression at 0.05 and 0.01 level,
respectively. (a) Upregulated genes and (b) downregulated genes.
Among the 22 validated unigenes, 20 and 8 genes were mapped to GO and
KEGG databases, respectively. For example, microtubule associated
protein 1B (MAP1B) gene, which is involved in microtubule assembly and
neurogenesis [[126]46,[127]47], was categorized into microtubule bundle
formation, microtubule associated complex, microtubule binding etc. GO
terms. Calcium channel, voltage-dependent, L type, alpha 1C subunit
(CACNA1C) gene encodes an important subunit in calcium channels, which
is related to heart disease and psychosis [[128]48]. In our study, this
gene was categorized into arrhythmogenic right ventricular
cardiomyopathy, calcium signalling pathway, cardiac muscle contraction,
Alzheimer's disease etc. pathways. Thus KEGG and GO term analysis could
well predict the possible function of unigenes and revealed potential
genes that play crucial roles in this study.
4. Conclusion
In conclusion, we revealed 1612 DEGs in ovariectomized goat muscle, and
24 and 11 DEGs involved in development and reproduction associated
pathways, respectively. These results provide valuable theoretical
basis for the research of molecular mechanisms of female animal
fattening and muscle development of ovariectomized animals.
Supplementary Material
Fupplementary file: The list of the primers which were used for the
qPCR analysis of 22 differently expressed genes
[129]rsos171415supp1.doc^ (85.5KB, doc)
Supplementary Material
Fupplementary file: Statistics data (including the data volume of raw
data and valid data, and the valid ration of reads) of the sequencing
result of five samples
[130]rsos171415supp2.doc^ (26KB, doc)
Supplementary Material
Fupplementary file: The list of the 1612 differently expressed genes
[131]rsos171415supp3.xls^ (517KB, xls)
Supplementary Material
Fupplementary file: The list of the genes which were mapped to Go terms
[132]rsos171415supp4.xls^ (164.5KB, xls)
Supplementary Material
Fupplementary file: The list of the genes which were enriched to
specific KEGG pathway
[133]rsos171415supp5.xls^ (64.5KB, xls)
Acknowledgements